Field of the Invention
The present invention relates to a method for detecting a target particle using an optical system of a confocal microscope or multi-photon microscope and the like capable of detecting light from a microregion in a solution.
Description of the Related Art
Due to progress made in the field of optical measurement technology in recent years, it has become possible to detect and measure feint light at the level of a single photon or single fluorescent molecule using the optical system of a confocal microscope and ultra-high-sensitivity photodetection technology capable of performing photon counting (detecting individual photons). Therefore, various devices or methods have been proposed that detect interactions between molecules such as biomolecules or coupling and dissociation reactions between molecules using such feint light measurement technology. In particular, according to a method such as fluorescence correlation spectroscopy (FCS) or fluorescence intensity distribution analysis (FIDA) that uses a technology for measuring fluorescence of a microregion using the optical system of a confocal microscope and photocounting technology, the sample required for measurement is only required to be at an extremely low concentration and in an extremely small amount in comparison with that used in the past, and the amount used for a single measurement is roughly only several tens of microliters. In addition, measurement time is shortened considerably, and measurement of a duration on the order of several seconds for a single measurement is repeated several times. Furthermore, the aforementioned microregion refers to a confocal region where laser light of a microscope is focused, and is referred to as confocal volume. Moreover, one example of an aspect of FCS consists of calculating the value of an autocorrelation function of fluorescence intensity in a system in which detected light intensity decreases when non-luminescent particles dispersed in a solution in which a large amount of luminescent substances have been dissolved have entered a confocal volume, followed by calculating the translational diffusion time of the non-luminescent particles in the confocal volume along with the average value of the number of particles retained therein (inverse-FCS, or iFCS) (see, for example, International Patent Publication No. WO2010/119098).
More recently, an optical analysis technology (scanning molecule counting method) employing a novel approach has been proposed that individually detects luminescent particles (particles that emit light by fluorescence, phosphorescence, chemiluminescence, bioluminescence or light scattering and the like) crossing a photodetection region in a sample solution in the form of a microregion while moving the location of the microregion using an optical system capable of detecting light from a microregion in a solution, such as the optical system of a confocal microscope or multi-photon microscope (see, for example, International Publication No. WO 2011/108369; International Publication No. WO 2011/108370; and International Publication No. WO 2011/108371). More specifically, the scanning molecule counting method is a technique that enables counting of luminescent particles, or acquiring information relating to concentration or number density of luminescent particles in a sample solution, by detecting light emitted from the luminescent particles in a photodetection region to individually detect each of the luminescent particles in a sample solution while moving the location of the photodetection region of an optical system of a confocal microscope or multi-photon microscope in the sample solution using that optical system.
Since the photodetection mechanism per se of the scanning molecule counting method is composed so as to detect light from a photodetection region of a confocal microscope or multi-photon microscope in the same manner as in the case of optical analysis technologies such as FIDA, the amount of sample solution may also be an extremely small amount (such as roughly several tens of microliters) and only a short measurement time is required in the same manner as optical analysis technologies such as FIDA. On the other hand, the scanning molecule counting method differs from FIDA and the like, which requires statistical processing involving calculation of fluctuations in fluorescence intensity and the like, in that such statistical processing is not carried out. Consequently, optical analysis technology employing the scanning molecule counting method can be applied to sample solutions in which the number density or concentration of particles is considerably lower than the level required by optical analysis technologies such as FIDA. In other words, by detecting a target particle (an observation target particle) in a sample solution labeled with a luminescent probe using the scanning molecule counting method, the status or properties of the target particle can be detected and analyzed even in the case the concentration or number density of the target particles in the sample solution is extremely low (see, for example, International Publication No. WO 2012/014778).